Cripto-1 localizes to dynamic and shed filopodia associated with cellular migration in glioblastoma cells

Cripto-1 is a protein participating in tissue orientation during embryogenesis but has also been implicated in a wide variety of cancers, such as colon, lung and breast cancer. Cripto-1 plays a role in the regulation of different pathways, including TGF-β/Smad and Wnt/β-catenin, which are highly associated with cell migration both during embryonal development and cancer progression. Little is known about the detailed subcellular localization of cripto-1 and how it participates in the directional movement of cells. In this study, the subcellular localization of cripto-1 in glioblastoma cells was investigated in vitro with high-resolution microscopy techniques. Cripto-1 was found to be localized to dynamic and shed filopodia and transported between cells through tunneling nanotubes. Our results connect the refined subcellular localization of cripto-1 to its functions in cellular orientation and migration.     

Twenty-four-hour variations in subcellular structures of rat type II alveolar epithelial cells

Summary Subcellular structures of type II alveolar epithelial cells in the rat lung were analyzed at six evenly spaced times over 24 h (light period: 06.00 h–18.00 h), using a morphometric technique. The cell volumes were maximal at 16.00 h and minimal at 08.00 h. The volume and surface densities of rough endoplasmic reticulum and mitochondria were low during the light period, and high during the dark period. Morphometric parameters of multivesicular bodies did not significantly fluctuate over 24 h, but they increased from 04.00 h to 08.00 h. The volume densities of lamellar bodies increased from 16.00 h to 20.00 h, and decreased from 00.00 h to 08.00 h. The change in numerical densities of lamellar bodies was inversely correlated to that in the volume densities. As shown by electron microscopy, small lamellar bodies predominated at 08.00 h, larger lamellar bodies increasing at 16.00h. Composite bodies often appeared at 08.00 h and 12.00 h. Type II cells thus appear to fluctuate, showing three phases over 24 h: formation, accumulation and secretion of lamellar bodies. In particular, it is noteworthy that the accumulation stage occurs during the resting phase of the rat, whereas the secretion stage occurs during its body-active phase.     

Subcellular Distribution of UDP-Galactose:Ceramide Galactosyltransferase in Rat Brain Oligodendroglia

Oligodendrocytes isolated from 18-19-day-old rat brain were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 100,000 g for 50 min in a gradient containing 0.8, 1.05, and 1.3 M sucrose. Three discrete bands were obtained at the interfaces 0.32-0.8 (F1), 0.8-1.05 (F2), and 1.05-1.3 M (F3). The distribution of UDP-galactose:ceramide galactosyltransferase (CgalT) activity in each fraction was measured using liposomes containing normal fatty acid-containing ceramides (NFA-CgalT activity) or 2-hydroxy fatty acid-containing ceramides (HFA-CgalT activity). Although detection of both CgalT activities was possible in all fractions, HFA-CgalT activity was enriched in F1 and F2 fractions, which also showed an enrichment of Golgi and endoplasmic reticulum markers, respectively. It is interesting that NFA-CgalT activity was significantly enriched in the F2 fraction. These results suggest that hydroxylated and nonhydroxylated galactocerebrosides may be synthesized at different intracellular locations.     

Subcellular Localization of Rat Brain Insulin Binding Sites

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, gamma-aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mitochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r = 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Generation of phosphatidylinositol 4,5-bisphosphate proceeds through an intracellular route in rat hepatocytes

The subcellular distribution in rat hepatocytes of enzymes participating in the entire generation cycle of phosphatidylinositol 4,5-bisphosphate, and phosphorylated intermediates of this pathway, has been examined by Nycodenz gradient centrifugation. Our results indicate that the synthesis of phosphatidylinositol takes place in the endoplasmic reticulum, and that its phosphorylation to phosphatidylinositol 4-phosphate occurs intracellularly in low-density membranes before translocation to the plasma membrane, where it is further phosphorylated to phosphatidylinositol 4,5-bisphosphate. The intracellular formation of PIP implies a vesicular transport to the plasma membrane.     

Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe

The subcellular localization of the enzyme invertase in Schizosaccharomyces pombe cells, both repressed and derepressed for synthesis of the enzyme, was studied. Most of the invertase was found to be located outside the plasma membrane and only a small percentage was found to be associated to membranes. A substantial portion of the external enzyme remained firmly bound to cell-wall material.All of the invertase recovered in soluble form from cellular extracts reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds, indicating the presence in the enzyme of a carbohydrate moiety which probably contains terminal mannosyl (or structurally related) and galactosyl residues.The possibility of the presence of two different forms of invertase in S. pombe was considered. An intracellular, soluble form of invertase, devoid of carbohydrate, similar to the small invertase of the budding yeast Saccharomyces cerevisiae, was not found in S. pombe. However, the Michaelis constant for sucrose of the enzyme present in repressed cells was smaller than that of the invertase synthesized under derepressing conditions, although this difference could also be the result of a different pattern of glycosylation of the invertase synthesized under different growth conditions.     

Long-Term Effect of Forskolin on the Activation of Adenylyl Cyclase in Astrocytes

Abstract: Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30–40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(βγ-imido)diphosphate, guanosine 5'- O -(3-thiotriphosphate) [GTP(S)], and AIF₄⁻ in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25–1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in G_sα, G_iα, or Gβ protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc⁻ cells, lacking G_sα protein. G_sα proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted G_sα protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via G_sα, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin.     

Molecular evolution of the HSP70 multigene family

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups. Correspondence to: E.A. Craig